Follicle stimulating hormone superagonists

ABSTRACT

The invention is directed toward a human glycoprotein hormone having at least one, two, three, four, or five basic amino acids in the α-subunit at positions selected from the group consisting of positions 11, 13, 14, 16, 17, and 20. The inventions is also directed to a human glycoprotein where at least one of the amino acids at position 58, 63, and 69 of the β-subunit of the human thyroid stimulating hormone are basic amino acids. The invention is further directed to a modified human glycoprotein hormone having increased activity over a wild-type human glycoprotein hormone, where the modified human glycoprotein comprises a basic amino acid substituted at a position corresponding to the same amino acid position in a non-human glycoprotein hormone having an increased activity over the wild-type human glycoprotein hormone. The invention is also directed to a method of constructing superactive nonchimeric analogs of human hormones comprising comparing the amino acid sequence of a more active homolog from another species to the human hormone, and selecting superactive analogs from the substituted human hormones. The invention is also directed to nucleic acids encoding the modified human glycoprotein hormones, vectors containing those nucleic acids, and host cells containing those vectors.

This application is a divisional of prior application Ser. No.09/185,408 filed Nov. 3, 1998 now U.S. Pat. No. 6,361,992 which is acontinuation of PCT/US96/06483 filed May 8, 1996 designating the UnitedStates of America and published in English.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to modified glycoprotein hormones.Specifically, this invention relates to modifications to a humanglycoprotein which create superagonist activity.

2. Background Art

Thyrotropin (thyroid-stimulating hormone, TSH) and the gonadotropinschorionic gonadotropin, (CG), lutropin (luteinizing hormone, LH), andfollitropin (follicle-stimulating hormone, FSH) comprise the family ofglycoprotein hormones. Each hormone is a heterodimer of twonon-covalently linked subunits: α and β. Within the same species, theamino acid sequence of the α-subunit is identical in all the hormones,whereas the sequence of the β-subunit is hormone specific. (Pierce, J.G. and Parsons, T. F. “Glycoprotein hormones: structure and function.”Ann. Rev. Biochem. 50:465–495 (1981)). The fact that the sequences ofthe subunits are highly conserved from fish to mammals implies thatthese hormones have evolved from a common ancestral protein (FontaineY-A. and Burzawa-Gerard, E. “Esquisse de l'evolution des hormonesgonadotopes et thyreotropes des vertebres.” Gen. Comp. Endocrinol.32:341–347 (1977)). Evolutionary changes of these hormones resulted incertain cases in modification of biological activity (Licht, P. et al.“Evolution of gonadotropin structure and function.” Rec. Progr. Horm.Res., 33:169–248 (1977) and Combarnous, Y. “Molecular basis of thespecificity of binding of glycoprotein hormones to their receptors.”Endocrine Rev. 13:670–691 (1992)), although, specific structuraldeterminants modulating biopotency have not been elucidated. Forexample, human thyroid stimulating hormone (hTSH) and bovine thyroidstimulating hormone (bTSH) share high homology in the α (70%) and β(89%) subunit sequence, but bTSH is 6–10 fold more potent than hTSH(Yamazaki, K. et al. “Potent thyrotropic activity of human chorionicgonadotropin variants in terms of ¹²⁵I incorporation and de novosynthesized thyroid hormone release in human thyroid follicles.” J.Clin. Endocrinol. Metab. 80:473–479 (1995)).

Glycoprotein hormones are crucial in certain therapies, such as in thetreatment of patients with thyroid carcinoma. (See, for example, Meier,C. A., et al., “Diagnostic use of Recombinant Human Thyrotropin inPatients with Thyroid Carcinoma (Phase I/II Study).” J. Clin.Endocrinol. Metabol. 78:22 (1994)). The potential use of human thyroidstimulating hormone (TSH) in the treatment of this disease has beenabandoned due to the potential transmission of Creutzfeldt-Jakobdisease. An alternative to the use of human TSH is the use of bovineTSH, but this approach is very limited since this hormone causesside-effects such as nausea, vomiting, local induration, urticaria, anda relatively high possibility of anaphylactic shock (Meier, C. A., etal.). The lack of bioconsistency of urinary gonadotropins and thelimited efficacy of recombinant glycoprotein hormones justify theirfurther replacement with more effective recombinant analogs. Therefore,there is a need for human-derived glycoprotein hormones as well asagonists of these hormones.

For example the administration of an agonist of the thyroid stimulatinghormone in a particular clinical situation such as thyroid carcinoma,will enhance the uptake of radioiodine into the carcinoma to treat thedisease. Agonists of the thyroid stimulating hormone will cause agreater amount of the radioiodine to be targeted to the carcinoma,thereby resulting in a more effective treatment. Alternatively,glycoprotein hormones used to induce ovulation can be replaced withsuperagonists. This will lower the required dose of the hormone whichcurrently is a major medical problem in fertility treatment.(Ben-Rafael, Z., et al. “Pharmacokinetics of follicle-stimulatinghormone: clinical significance.” Fertility and Sterility 63:689 (1995)).Where the use of wild-type follicle stimulating hormone has led tohyperstimulation and higher rates of multiple pregnancies and abortions,apparently by a high number of hormone molecules stimulating manyfollicles, a superagonist of follicle-stimulating hormone can beadministered to treat the infertility. The use of an agonist of thismodified hormone can result in a lower frequency of stimulation ofmultiple follicles since a lower number of hormone molecules can beadministered to achieve the desired result.

The present invention provides, for the first time, specific amino acidsubstitutions in human glycoprotein hormones which results in humanglycoprotein hormone analogs that show a major increase in both in vitroand in vivo bioactivity.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, provides ahuman glycoprotein hormone comprising at least three basic amino acidsin the α-subunit at positions selected from the group consisting ofpositions 11, 13, 14, 16, 17 and 20.

The invention further provides a human glycoprotein hormone comprisingat least one basic amino acid in the α-subunit at positions selectedfrom the group consisting of positions 11, 13, 14, 16, 17 and 20.

In another aspect, the invention provides a modified human glycoproteinhormone having increased activity over a wild-type human glycoprotein,wherein the modified human hormone comprises a basic amino acidsubstituted at a position corresponding to the same amino acid positionin a non-human glycoprotein hormone having an increased activity overthe wild-type human glycoprotein.

In another aspect, the invention provides a method of treating acondition associated with a glycoprotein hormone activity in a subjectcomprising administering a therapeutic amount of the glycoproteinhormone of the present invention to the patient.

In another aspect, the invention provides a method of constructingsuperactive nonchimeric analogs of human hormones comprising comparingthe amino acid sequence of a more active homolog from another species tothe human hormone, substituting amino acids in the human hormone withthe corresponding amino acids from the other species, determining theactivity of the substituted human hormone, and selecting superactiveanalogs from the substituted human hormones.

In yet another aspect, the present invention provides nucleic acidswhich encode the modified glycoprotein hormones.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the relevant primary sequences of theα-subunit from 27 different species (a). Alignment of the subunitsequences obtained from sequencing of PCR amplified fragment of genomicDNA in chimpanzee, orangutan, gibbon and baboon (underlined), receivedfrom GeneBank, SWISS-PROT and PDB databank were made. The numbering ofthe sequences corresponds to that of human α-subunit sequence. Dashes( - - - ) indicate amino acid residues which are identical to those ofthe human α-subunit. Conserved among different species lysine residuesare bolded. The primate sequences determined in this study areunderlined. The human, chimpanzee and orangutan α-subunit sequences arethe only sequences without basic amino acids in this region, despite therelatively high degree of similarity in diverse vertebrate species. (b)Mutations of human sequence made in this region included introduction ofsingle and multiple Lys residues present in all non-human mammaliansequences. Additionally, alanine mutagenesis of residues 13, 16 and 20was used to study the role of Gln13, Pro16 and Gln20.

FIG. 2 shows the bioactivities and receptor binding activities of themost potent hTSH analogs: (a, b) cAMP stimulation in CHO-JP09 cells.Data represent the mean ±SEM of triplicate determinations from arepresentative experiment repeated three (a) and two (b) times. (c, d)Receptor-binding activities to CHO-JP09 cells. The same mutants testedas in the FIG. 2 a and FIG. 2 b respectively. Values are the mean ±SEMof quadruplicate determinations from one experiment, repeated two times.(e) Thymidine uptake stimulation in FRTL-5 cells. Values are the mean±SEM of quadruplicate determinations from one experiment, repeated twotimes. (f) Stimulation of T₄ secretion in mice. Each data pointrepresents the mean ±SEM of values from 4–5 animals of a representativeexperiment repeated two times. (g) cAMP stimulation in CHO-hTSH cells.Data represent the mean ±SEM of 3–4 determinations from a representativeexperiment repeated 3 times.(h) Receptor-binding activities in CHO-JP09cells. Data represent the mean ±SEM of 3–4 determinations from arepresentative experiment repeated 3 times.(i) Stimulation of T₄secretion in mice. Each data point represents the mean ±SEM of valuesfrom 4–5 animals of a representative experiment repeated two times.

FIG. 3 shows the bioactivities and receptor binding activities of themost potent hCG analogs. Progesterone production stimulation (a) andreceptor binding assay (b) in MA-10 cells. Data represent the mean ±SEMof triplicate determinations from a representative experiment repeatedthree times. The relative maximal production levels of progesterone arepresented in the Table II as % obtained with WT-hCG. cAMP stimulation(c) and receptor binding assay (d) in COS-7 cells expressing hLHreceptor. Data represent the mean ±SEM of triplicate determinations froma representative experiment repeated two times.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Example included therein and to the Figures and theirprevious and following description.

Before the present compounds, compositions, and methods are disclosedand described, it is to be understood that this invention is not limitedto specific hormones, specific subjects, i.e. humans as well asnon-human mammals, specific amino acids, specific clinical conditions,specific analogs, or specific methods, as such may, of course, vary, andthe numerous modifications and variations therein will be apparent tothose skilled in the art. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

As used in the specification and in the claims, “a” can mean one ormore, depending upon the context in which it is used. Thus, for example,reference to “a human glycoprotein hormone” means that at least onehuman glycoprotein hormone is utilized.

In one aspect, the invention provides a human glycoprotein hormonecomprising at least three basic amino acids in the α-subunit atpositions selected from the group consisting of positions 11, 13, 14,16, 17 and 20.

The invention further provides a human glycoprotein hormone comprisingat least one basic amino acid in the α-subunit at positions selectedfrom the group consisting of positions 11, 13, 14, 16, 17 and 20.

In another aspect, the invention provides a modified human glycoproteinhormone having increased activity over a wild-type human glycoprotein,wherein the modified human hormone comprises a basic amino acidsubstituted at a position corresponding to the same amino acid positionin a non-human glycoprotein hormone having an increased activity overthe wild-type human glycoprotein.

In another aspect, the invention provides a method of treating acondition associated with a glycoprotein hormone activity in a subjectcomprising administering a therapeutic amount of the glycoproteinhormone of the present invention to the patient.

In another aspect, the invention provides a method of assistingreproduction in a subject comprising administering an assisting amountof the glycoprotein hormone of the present invention.

In another aspect, the invention provides a method of constructingsuperactive nonchimeric analogs of human hormones comprising comparingthe amino acid sequence of a more active homolog from another species tothe human hormone, substituting amino acids in the human hormone withthe corresponding amino acids from the other species, determining theactivity of the substituted human hormone, and selecting superactiveanalogs from the substituted human hormones.

By “human” glycoprotein hormone is meant that the number of amino acidsubstitutions made in the wild-type sequence does not exceed one-halfthe number of amino acid differences at corresponding positions in thecorresponding polypeptide hormones between human and another species.Thus, the modified polypeptide hormone would be considered more like thewild-type polypeptide hormone of the human than the correspondingpolypeptide hormone from the non-human species from which the amino acidsubstitutions are derived, based on the amino acid coding sequence. Forexample, if there were a total of 20 amino acid differences atcorresponding positions in corresponding glycoprotein hormones between ahuman glycoprotein and a bovine glycoprotein hormone, a “human”glycoprotein hormone would be a modified wild-type human hormone whichcontains 10 or fewer amino acid substitutions within its amino acidsequence which are homologous to the corresponding amino acids in thebovine amino acid sequence. More specifically, the thyroid stimulatinghormone, as set forth in the Examples contained herein, would beconsidered “human” if 20 or more of the 40 total amino acid differencesbetween the α- and β-subunits of the human and the bovine homologs arehomologous to the amino acid at the corresponding position in the humanthyroid stimulating hormone.

Naturally, because of the risk of an adverse immune response to theadministration of the modified glycoprotein hormone where the recipientof the modified glycoprotein hormone is a human, the modifiedglycoprotein hormone is preferably homologous to the human amino acidsequence to the greatest extent possible without an unacceptable loss inthe superagonist activity. Alternatively, where the subject beingadministered the modified glycoprotein is non-human, the modifiedglycoprotein hormone is preferably homologous to the specific non-humanamino acid sequence to the greatest extent possible without anunacceptable loss in the superagonist activity. Thus, in a preferredembodiment of the present invention, in modifying a wild-typeglycoprotein to construct a modified glycoprotein with a superagonistactivity by substituting specific amino acids, the substituted aminoacids which do not increase agonist activity number 10 or less,especially 9, 8, 7, 6, 5, 4, 3, and 2 or zero.

Likewise, by “nonchimeric” is meant that the number of aminosubstitutions does not exceed one-half the number of amino aciddifferences at corresponding positions in the corresponding polypeptidehormones between species, such that the modified polypeptide hormonewould be considered more like the wild-type polypeptide hormone of thespecies being modified than the corresponding polypeptide hormone fromthe species from which the amino acid substitutions are derived, basedon the amino acid coding sequence.

In yet another aspect, the present invention provides nucleic acidswhich encode the modified glycoprotein hormones.

Glycoprotein hormones comprise a family of hormones which arestructurally related heterodimers consisting of a species-commonα-subunit and a distinct β-subunit that confers the biologicalspecificity for each hormone. For a general review of glycoproteinhormones, see Pierce, J. G. et al., “Glycoprotein hormones: structureand function.” Ann. Rev. Biochem. 50:465–495 (1981), see alsoCombarnous, Y. “Molecular basis of the specificity of binding ofglycoprotein hormones to their receptors.” Endocrine Rev. 13:670–691(1992) This family of hormones includes chorionic gonadotropin (CG),lutropin (luteinizing hormone, LH), follitropin (follicle-stimulatinghormone, FSH), and thyrotropin (thyroid-stimulating hormone, TSH). Eachof these glycoprotein hormones with at least one basic amino acid in theα-subunit at positions selected from the group consisting of positions11, 13, 14, 16, 17, and 20, is provided by the present invention.

Basic amino acids comprise the amino acids lysine, arginine, andhistidine, and any other basic amino acid which may be a modification toany of these three amino acids, synthetic basic amino acids not normallyfound in nature, or any other amino acid which is positively charged ata neutral pH.

The glycoprotein hormones provided for by the present invention may beobtained in any number of ways. For example, a DNA molecule encoding aglycoprotein hormone can be isolated from the organism in which it isnormally found. For example, a genomic DNA or cDNA library can beconstructed and screened for the presence of the nucleic acid ofinterest. Methods of constructing and screening such libraries are wellknown in the art and kits for performing the construction and screeningsteps are commercially available (for example, Stratagene CloningSystems, La Jolla, Calif.). Once isolated, the nucleic acid can bedirectly cloned into an appropriate vector, or if necessary, be modifiedto facilitate the subsequent cloning steps. Such modification steps areroutine, an example of which is the addition of oligonucleotide linkerswhich contain restriction sites to the termini of the nucleic acid.General methods are set forth in Sambrook et al., “Molecular Cloning, aLaboratory Manual,” Cold Spring Harbor Laboratory Press (1989).

Once the nucleic acid sequence of the desired glycoprotein hormone isobtained, basic amino acids can be positioned at any particular aminoacid positions by techniques well known in the art. For example, PCRprimers can be designed which span the amino acid position or positionsand which can substitute a basic amino acid for a non-basic amino acid.Then a nucleic acid can be amplified and inserted into the wild-typeglycoprotein hormone coding sequence in order to obtain any of a numberof possible combinations of basic amino acids at any position of theglycoprotein hormone. Alternatively, one skilled in the art canintroduce specific mutations at any point in a particular nucleic acidsequence through techniques for point mutagenesis. General methods areset forth in Smith, M “In vitro mutagenesis” Ann. Rev. Gen., 19:423–462(1985) and Zoller, M. J. “New molecular biology methods for proteinengineering” Curr. Opin. Struct. Biol., 1:605–610 (1991).

Another example of a method of obtaining a DNA molecule encoding aspecific glycoprotein hormone is to synthesize a recombinant DNAmolecule which encodes the glycoprotein hormone. For example,oligonucleotide synthesis procedures are routine in the art andoligonucleotides coding for a particular protein region are readilyobtainable through automated DNA synthesis. A nucleic acid for onestrand of a double-stranded molecule can be synthesized and hybridizedto its complementary strand. One can design these oligonucleotides suchthat the resulting double-stranded molecule has either internalrestriction sites or appropriate 5′ or 3′ overhangs at the termini forcloning into an appropriate vector. Double-stranded molecules coding forrelatively large proteins can readily be synthesized by firstconstructing several different double-stranded molecules that code forparticular regions of the protein, followed by ligating these DNAmolecules together. For example, Cunningham, et al., “Receptor andAntibody Epitopes in Human Growth Hormone Identified by Homolog-ScanningMutagenesis,” Science, 243:1330–1336 (1989), have constructed asynthetic gene encoding the human growth hormone gene by firstconstructing overlapping and complementary synthetic oligonucleotidesand ligating these fragments together. See also, Ferretti, et al., Proc.Nat. Acad. Sci. 82:599–603 (1986), wherein synthesis of a 1057 base pairsynthetic bovine rhodopsin gene from synthetic oligonucleotides isdisclosed. By constructing a glycoprotein hormone in this manner, oneskilled in the art can readily obtain any particular glycoproteinhormone with basic amino acids at any particular position or positionsof either the α-subunit, the β-subunit, or both. See also, U.S. Pat. No.5,503,995 which describes an enzyme template reaction method of makingsynthetic genes. Techniques such as this are routine in the art and arewell documented. DNA fragments encoding glycoprotein hormones can thenbe expressed in vivo or in vitro as discussed below.

Once a nucleic acid encoding a particular glycoprotein hormone ofinterest, or a region of that nucleic acid, is constructed, modified, orisolated, that nucleic acid can then be cloned into an appropriatevector, which can direct the in vivo or in vitro synthesis of thatwild-type and/or modified glycoprotein hormone. The vector iscontemplated to have the necessary functional elements that direct andregulate transcription of the inserted gene, or hybrid gene. Thesefunctional elements include, but are not limited to, a promoter, regionsupstream or downstream of the promoter, such as enhancers that mayregulate the transcriptional activity of the promoter, an origin ofreplication, appropriate restriction sites to facilitate cloning ofinserts adjacent to the promoter, antibiotic resistance genes or othermarkers which can serve to select for cells containing the vector or thevector containing the insert, RNA splice junctions, a transcriptiontermination region, or any other region which may serve to facilitatethe expression of the inserted gene or hybrid gene. (See generally,Sambrook et al.).

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art which are useful for the expressionof the nucleic acid insert. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary, an amino terminalmethionine can be provided by insertion of a Met codon 5′ and in-framewith the downstream nucleic acid insert. Also, the carboxy-terminalextension of the nucleic acid insert can be removed using standardoligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion systems exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF″-1 gene) isroutinely used to direct protein secretion from yeast. (Brake, et al.,“∝-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins inSaccharomyces cerevisiae.” Proc. Nat. Acad. Sci., 81:4642–4646 (1984)).The leader region of pre-pro-alpha-factor contains a signal peptide anda pro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene: this enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. Thenucleic acid coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The nucleic acidcoding sequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thenucleic acid coding sequences can be fused to a second protein codingsequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.Efficient post translational glycosolation and expression of recombinantproteins can also be achieved in Baculovirus systems.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures, andsecretion of active protein. Vectors useful for the expression of activeproteins in mammalian cells are characterized by insertion of theprotein coding sequence between a strong viral promoter and apolyadenylation signal. The vectors can contain genes conferringhygromycin resistance, gentamicin resistance, or other genes orphenotypes suitable for use as selectable markers, or methotrexateresistance for gene amplification. The chimeric protein coding sequencecan be introduced into a Chinese hamster ovary (CHO) cell line using amethotrexate resistance-encoding vector, or other cell lines usingsuitable selection markers. Presence of the vector DNA in transformedcells can be confirmed by Southern blot analysis. Production of RNAcorresponding to the insert coding sequence can be confirmed by Northernblot analysis. A number of other suitable host cell lines capable ofsecreting intact human proteins have been developed in the art, andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkatcells, etc. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation is commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofectin mediated transfection or electroporation maybe used for othercellular hosts.

Alternative vectors for the expression of genes in mammalian cells,those similar to those developed for the expression of humangamma-interferon, tissue plasminogen activator, clotting Factor VIII,hepatitis B virus surface antigen, protease Nexinl, and eosinophil majorbasic protein, can be employed. Further, the vector can include CMVpromoter sequences and a polyadenylation signal available for expressionof inserted nucleic acids in mammalian cells (such as COS-7).

Expression of the gene or hybrid gene can be by either in vivo or invitro. In vivo synthesis comprises transforming prokaryotic oreukaryotic cells that can serve as host cells for the vector. An exampleof modified glycoprotein hormones inserted into a prokaryotic expressionvector is given in the Example section contained herein.

Alternatively, expression of the gene can occur in an in vitroexpression system. For example, in vitro transcription systems arecommercially available which are routinely used to synthesize relativelylarge amounts of mRNA. In such in vitro transcription systems, thenucleic acid encoding the glycoprotein hormone would be cloned into anexpression vector adjacent to a transcription promoter. For example, theBluescript II cloning and expression vectors contain multiple cloningsites which are flanked by strong prokaryotic transcription promoters.(Stratagene Cloning Systems, La Jolla, Calif.). Kits are available whichcontain all the necessary reagents for in vitro synthesis of an RNA froma DNA template such as the Bluescript vectors. (Stratagene CloningSystems, La Jolla, Calif.). RNA produced in vitro by a system such asthis can then be translated in vitro to produce the desired glycoproteinhormone. (Stratagene Cloning Systems, La Jolla, Calif.).

Another method of producing a glycoprotein hormone is to link twopeptides or polypeptides together by protein chemistry techniques. Forexample, peptides or polypeptides can be chemically synthesized usingcurrently available laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to a hybrid glycoprotein hormone can be synthesized bystandard chemical reactions. For example, a peptide or polypeptide canbe synthesized and not cleaved from its synthesis resin whereas theother fragment of a hybrid peptide can be synthesized and subsequentlycleaved from the resin, thereby exposing a terminal group which isfunctionally blocked on the other fragment. By peptide condensationreactions, these two fragments can be covalently joined via a peptidebond at their carboxyl and amino termini, respectively, to form a hybridpeptide. (Grant, G. A., “Synthetic Peptides: A User Guide,” W.H. Freemanand Co., N.Y. (1992) and Bodansky, M. and Trost, B., Ed., “Principles ofPeptide Synthesis,” Springer-Verlag Inc., N.Y. (1993)). Alternatively,the peptide or polypeptide can by independently synthesized in vivo asdescribed above. Once isolated, these independent peptides orpolypeptides may be linked to form a glycoprotein hormone via similarpeptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentscan allow relatively short peptide fragments to be joined to producelarger peptide fragments, polypeptides or whole protein domains(Abrahmsen, L., et al., Biochemistry, 30:4151 (1991)). Alternatively,native chemical ligation of synthetic peptides can be utilized tosynthetically construct large peptides or polypeptides from shorterpeptide fragments. This method consists of a two step chemical reaction(Dawson, et al., “Synthesis of Proteins by Native Chemical Ligation,”Science, 266:776–779 (1994)). The first step is the chemoselectivereaction of an unprotected synthetic peptide-∝-thioester with anotherunprotected peptide segment containing an amino-terminal Cys residue togive a thioester-linked intermediate as the initial covalent product.Without a change in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Clark-Lewis, I., et al.,FEBS Lett., 307:97 (1987), Clark-Lewis, I., et al., J.Biol.Chem.,269:16075 (1994), Clark-Lewis, L, et al., Biochemistry, 30:3128 (1991),and Rajarathnam, K, et al., Biochemistry, 29:1689 (1994)).

Alternatively, unprotected peptide segments can be chemically linkedwhere the bond formed between the peptide segments as a result of thechemical ligation is an unnatural (non-peptide) bond (Schnolzer, M, etal., Science, 256:221 (1992)). This technique has been used tosynthesize analogs of protein domains as well as large amounts ofrelatively pure proteins with full biological activity (deLisle Milton,R. C., et al., “Techniques in Protein Chemistry IV,” Academic Press, NewYork, pp. 257–267 (1992)).

The invention also provides fragments of modified glycoprotein hormoneswhich have either superagonist or antagonist activity. The polypeptidefragments of the present invention can be recombinant proteins obtainedby cloning nucleic acids encoding the polypeptide in an expressionsystem capable of producing the polypeptide fragments thereof. Forexample, one can determine the active domain of a glycoprotein hormonewhich, together with a β-subunit, can interact with a glycoproteinhormone receptor and cause a biological effect associated with theglycoprotein hormone. In one example, amino acids found to notcontribute to either the activity or the binding specificity or affinityof the glycprotein hormone can be deleted without a loss in therespective activity.

For example, amino or carboxy-terminal amino acids can be sequentiallyremoved from either the native or the modified glycoprotein hormone andthe respective activity tested in one of many available assays. Inanother example, a fragment of a modified glycoprotein can comprise amodified hormone wherein at least one amino acid has been substitutedfor the naturally occurring amino acid at specific positions in eitherthe α or the β-subunit, and a portion of either amino terminal orcarboxy terminal amino acids, or even an internal region of the hormone,has been replaced with a polypeptide fragment or other moiety, such asbiotin, which can facilitate in the purification of the modifiedglycoprotein hormone. For example, a modified glycoprotein can be fusedto a maltose binding protein, through either peptide chemistry ofcloning the respective nucleic acids encoding the two polypeptidefragments into an expression vector such that the expression of thecoding region results in a hybrid polypeptide. The hybrid polypeptidecan be affinity purified by passing it over an amylose affinity column,and the modified glycoprotein can then be separated from the maltosebinding region by cleaving the hybrid polypeptide with the specificprotease factor Xa. (See, for example, New England Biolabs ProductCatalog, 1996, pg. 164:).

Active fragments of a glycoprotein hormone can also be synthesizeddirectly or obtained by chemical or mechanical disruption of largerglycoprotein hormone. An active fragment is defined as an amino acidsequence of at least about 5 consecutive amino acids derived from thenaturally occurring amino acid sequence, which has the relevantactivity, e.g., binding or regulatory activity.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the peptide is not significantly altered orimpaired compared to the modified glycoprotein hormone. Thesemodifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, etc. In any case, the peptide must possess a bioactiveproperty, such as binding activity, regulation of binding at the bindingdomain, etc. Functional or active regions of the glycoprotein hormonemay be identified by mutagenesis of a specific region of the hormone,followed by expression and testing of the expressed polypeptide. Suchmethods are readily apparent to a skilled practitioner in the art andcan include site-specific mutagenesis of the nucleic acid encoding thereceptor. (Zoller, M. J. et al.).

In one embodiment of the present invention, the human glycoproteinhormone comprises at least one basic amino acid in the α-subunit at theposition selected from the group consisting of positions 11, 13, 14, 16,17, and 20. In one embodiment, the human glycoprotein hormone has abasic amino acid at position 11. In another embodiment, the humanglycoprotein hormone has a basic amino acid at position 13. In anotherembodiment, the human glycoprotein hormone has a basic amino acid atposition 14. In another embodiment, the human glycoprotein hormone has abasic amino acid at position 16. In another embodiment, the humanglycoprotein hormone has a basic amino acid at position 17. In anotherembodiment, the human glycoprotein hormone has a basic amino acid atposition 20. In another embodiment of the present invention, the basicamino acid at position 11, 13, 14, 16, and 20 is lysine. In yet anotherembodiment of the present invention, the basic amino acid at position 17is arginine.

The present invention also provides for a human glycoprotein hormonewith basic amino acids in the α-subunit in all combinations of any twopositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20. For example, basic amino acids may be present atpositions 11 and 13, or positions 11, and 14, or positions 11 and 16, orpositions 11 and 17, or positions 11 and 20, or positions 13 and 14, orpositions 13 and 17, or positions 14 and 16, or positions 14 and 17, orpositions 14 and 20, or positions 16 and 17, or positions 17 and 20. Inone embodiment of the present invention, the human glycoprotein hormonehas basic amino acids at position 16 and 13. In another embodiment ofthe present invention, the human glycoprotein hormone has basic aminoacids at positions 20 and 13. In yet another embodiment, the humanglycoprotein hormone has basic amino acids at positions 16 and 20.

The present invention also provides for a human glycoprotein hormonewith basic amino acids in the α-subunit in all combinations of any threepositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20. For example, basic amino acids may be present atpositions 11, 13, and 14, or positions 11, 13, and 16, or positions 11,13, and 17, or positions 11, 13, and 20, or positions 11, 14, and 16, orpositions 11, 14, and 17, or positions 11, 14, and 20, or positions 11,16, and 17, or positions 11, 16, and 20, or positions 11, 17, and 20, orpositions 13, 14, and 16, or positions 13, 14, and 17, or positions 13,14, and 20, or positions 13, 16, and 17, or positions 13, 17, and 20, orpositions 14, 16, and 17, or positions 14, 16, and 20, or positions 14,17, and 20, or positions 16, 17, and 20. In a preferred embodiment ofthe present invention, the human glycoprotein hormone has basic aminoacids at positions 13, 16, and 20. In another embodiment of the presentinvention, the hormone is thyroid stimulating hormone. In anotherembodiment of the present invention, the hormone is follicle-stimulatinghormone. In another embodiment of the present invention, the hormone isluteinizing hormone. In another embodiment of the present invention, thehormone is chorionic gonadotropin. In yet another embodiment of thepresent invention, the basic amino acids at any three positions selectedfrom the group consisting of positions 11, 13, 14, 16, 17, and 20, arelysine.

The present invention also provides for a human thyroid stimulatinghormone with at least three basic amino acids in the α-subunit atpositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20, where the thyroid stimulating hormone also has a basicamino acid in at least one position selected from the group consistingof positions 58, 63, and 69 of the β-subunit. In one embodiment of thepresent invention, the thyroid stimulating hormone has a basic aminoacid in at position 58 of the β-subunit. In another embodiment of thepresent invention, the thyroid stimulating hormone has a basic aminoacid in at position 63 of the β-subunit. In another embodiment of thepresent invention, the thyroid stimulating hormone has a basic aminoacid in at position 69 of the β-subunit. In another embodiment of thepresent invention, the thyroid stimulating hormone has a basic aminoacid in each of positions 58, 63, and 69 of the β-subunit. In yetanother embodiment of the present invention, the basic amino acid in atleast one position selected from the group consisting of positions 58,63, and 69 of the β-subunit is arginine.

The present invention also provides for a human glycoprotein hormonewith basic amino acids in the α-subunit in all combinations of any fourpositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20. One skilled in the art will readily determine thepossible combinations available. In one embodiment, the humanglycoprotein hormone has basic amino acids at positions 11, 13, 16, and20. In another embodiment, the human glycoprotein hormone has basicamino acids at positions 11, 13, 17, and 20. In another embodiment, thehuman glycoprotein hormone has basic amino acids at positions 13, 14,17, and 20. In a preferred embodiment, the human glycoprotein hormonehas basic amino acids at positions 13, 14, 16, and 20. In yet anotherembodiment of the present invention, the basic amino acids at any fourpositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20, are lysine.

The present invention also provides for a human glycoprotein hormonewith basic amino acids in the α-subunit in all combinations of any fivepositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20. One skilled in the art will readily determine thepossible combinations available. In one embodiment, the humanglycoprotein hormone has basic amino acids at positions 13, 14, 16, 17,and 20. In another embodiment, the human glycoprotein hormone has basicamino acids at positions 11, 13, 14, 16, and 20. In yet anotherembodiment of the present invention, the basic amino acids at any fivepositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20, are selected from the group consisting of lysine andarginine.

The present invention also provides for a human glycoprotein hormonewith basic amino acids in the α-subunit in all six of positions 11, 13,14, 16, 17, and 20.

In another aspect, the present invention provides a human glycoproteinhormone with a basic amino acid in the α-subunit in at least oneposition selected from the group consisting of positions 11, 13, 14, 16,17, and 20, wherein the hormone is human thyroid stimulating hormone andthere is a basic amino acid in at least one position selected from thegroup consisting of positions 58, 63, and 69 of the β-subunit. In oneembodiment of the present invention, the human glycoprotein hormone hasa basic amino acid at position 58 of the β-subunit of the human thyroidstimulating hormone. In another embodiment of the present invention, thehuman glycoprotein hormone has a basic amino acid at position 63 of theβ-subunit of the human thyroid stimulating hormone. In a preferredembodiment of the present invention, the human glycoprotein hormone hasa basic amino acid at position 69 of the β-subunit of the human thyroidstimulating hormone. In another embodiment of the present invention, thehuman glycoprotein hormone has basic amino acids at position 58, 63, and69 of the β-subunit of the human thyroid stimulating hormone. In yetanother embodiment of the present invention, the basic amino acid at theposition selected from the group consisting of positions 58, 63, and 69is arginine.

In another aspect, the present invention provides a humanfollicle-stimulating hormone, a human luteinizing hormone, or a humanchorionic gonadotropin glycoprotein hormone, wherein the hormonecomprises a basic amino acid in at least one position selected from thegroup consisting of positions in the β-subunit of any of theglycoprotein hormones, corresponding to positions 58, 63, and 69 of theβ-subunit of the human thyroid stimulating hormone. This approachapplies equally to non-humans as well. For example, the β-subunit aminoacid sequences of two bovine glycoprotein hormones can be compared andsubstitutions made to any of the subunits based on the sequencedifferences.

One skilled in the art can readily determine which sites of theβ-subunits of the other glycoprotein hormones correspond to sites 58,63, and 69 of the β-subunit of the human thyroid stimulating hormone.For example, see Ward, et al., In: Bellet, D and Bidard, J. M. (eds)“Structure-function relationships of gonadotropins” Serono SymposiumPublications, Raven Press, New York, 65:1–19 (1990), where the aminoacid sequences of 26 various glycoprotein hormone β-subunits are alignedand compared. Therefore, one skilled in the art can readily substitutenon-basic amino acids at these sites of the other glycoprotein hormonesfor basic amino acids.

Similarly, the present invention provides for any human glycoprotein,wherein the hormone comprises a basic amino acid in at least oneposition selected from the group consisting of positions in theβ-subunit of a glycoprotein hormone corresponding to the same positionsin any of the other human glycoprotein hormones. For example, the aminoacid sequence of the β-subunits of the human luteinizing hormone and thehuman chorionic gonadotropin hormone can be compared and amino acidsubstitutions made at selected sites in either of these glycoptoteinhormones based on the amino acid differences between the two β-subunits.This approach also applies equally to non-humans as well.

The present invention also provides a modified human glycoproteinhormone having increased activity over a wild-type human glycoprotein,wherein the modified human hormone comprises a basic amino acidsubstituted at a position corresponding to the same amino acid positionin a non-human glycoprotein hormone having an increased activity overthe wild-type human glycoprotein.

The non-human glycoprotein hormone having an increased activity over thewild-type human glycoprotein can be any non-human species. For example,the non-human species can be bovine. See, for example, Benua, R. S., etal “An 18 year study of the use of beef thyrotropin to increase I-131uptake in metastatic thyroid cancer.” J. Nucl. Med. 5:796–801 (1964) andHershman, J. M., et al. Serum thyrotropin (TSH) levels after thyroidablation compared with TSH levels after exogenous bovine TSH:implications for I-131 treatment of thyroid carcinoma.” J. Clin.Endocrinol. Metab. 34:814–818 (1972). Alternatively, the non-humanspecies can be equine, porcine, ovine, and the like. In the Examplecontained herein, the sequence of the 10–21 amino acid region of 27species is set forth.

The present invention also provides a modified glycoprotein hormonehaving increased activity over a wild-type glycoprotein hormone from thesame species, wherein the modified glycoprotein hormone comprises abasic amino acid substituted at a position corresponding to the sameamino acid position in a glycoprotein hormone from another specieshaving an increased activity over the wild-type glycoprotein hormone.Therefore the glycoprotein being modified to increase its activity canbe from a non-human species. For example, one can compare porcineglycoprotein hormones to bovine glycoprotein hormones, design porcineglycoprotein hormones with amino acid substitutions at positions wherethe porcine and the bovine sequences are different, construct porcineglycoprotein hormones with the selected changes, and administer themodified porcine glycoprotein hormone to porcine animals. Alternatively,the glycoprotein hormone being modified can be bovine.

The present invention also provides a modified glycoprotein hormonehaving increased activity over the wild-type glycoprotein hormone fromthe same species, wherein the modified glycoprotein hormone comprises abasic amino acid substituted at a position corresponding to the sameamino acid position in a different glycoprotein hormone from the samespecies having an increased activity over the wild-type glycoproteinhormone. For example, the β-subunits of human thyroid-stimulatinghormone and human chorionic gonadotropin can be compared and amino acidsubstitutions to either of these β-subunits can be made based on anysequence divergence. Naturally, only those changes which generallyincrease or decrease the activity of the modified glycoprotein hormoneare contemplated since the hormone receptor specificity will still needto be retained. An example of such a β-subunit modification is set forthin the Examples contained herein, where basic amino acids weresubstituted at positions 58 and 63 of the human thyroid stimulatinghormone based on sequence comparison between the human thyroidstimulating hormone and the human chorionic gonadotropin hormone.

Modification refers to the substitution of a non-basic amino acid at anyparticular position or positions of the wild-type glycoprotein with abasic amino acid. In a presently preferred embodiment of the presentinvention, these modifications comprise the substitution of lysine for anon-basic amino acid.

The effect of the modification or modifications to the wild-typeglycoprotein hormone can be ascertained in any number of ways. Forexample, cyclic AMP (cAMP) production in cells transfected with themodified glycoprotein can be measured and compared to the cAMPproduction of similar cells transfected with the wild-type glycoproteinhormone. Alternatively, progesterone production in cells transfectedwith the modified glycoprotein can be measured and compared to theprogesterone production of similar cells transfected with the wild-typeglycoprotein hormone. Alternatively, the activity of a modifiedglycoprotein hormone can be determined from receptor binding assays,from thymidine uptake assays, or from T₄ secretion assays. Specificexamples of such assays for determining the activity of modifiedglycoprotein hormones is set forth in the Example section containedherein. One skilled in the art can readily determine any appropriateassay to employ to determine the activity of either a wild-type or amodified glycoprotein hormone.

In one embodiment of the present invention, the modified glycoproteinhormone has an activity which is increased over the activity of the wildtype glycoprotein hormone by at least 3 fold. This increased activitycan be assessed by any of the techniques mentioned above and describedin the Example contained herein, or in any other appropriate assay asreadily determined by one skilled in the art. The increased activitydoes not have to be consistent from assay to assay, or from cell line tocell line, as these of course, will vary. For example, and as set forthin the Example contained herein, the relative potency of the P16Kmutation in the α-subunit of the human glycoprotein hormone compared tothe activity of the wild type glycoprotein hormone in a cAMP assay wasapproximately 6.4 fold higher. In the progesterone release assay,however, the difference between the same mutant and the wild-typeglycoprotein hormone was approximately 3.4 fold in potency and 1.6 foldin Vmax. This specific modification demonstrates at least a 3 foldincrease in activity in at least one assay, and therefore represents aglycoprotein hormone with at least a 3 fold increase in activity.

To modify additional amino acid positions, glycoprotein hormonesequences from human and non-humans can be aligned using standardcomputer software programs such as DNASIS (Hitachi Software EngineeringCo. Ltd.). The amino acid residues that differ between the human and thenon-human glycoprotein hormone can then be substituted using one of theabove-mentioned techniques, and the resultant glycoprotein hormoneassayed for its activity using one of the above-mentioned assays.

The subject being treated or administered a modified glycoproteinhormone can be a human or any non-human mammal. For example, themodified glycoprotein hormone superagonists may be used in thesuperovulation of bovine animals by administering these glycoproteinhormones to those bovine animals.

The methods used in substituting a basic amino acid for a non-basicamino acid at any particular position or positions can also be used todesign glycoprotein hormone antagonists. By making specificsubstitutions and monitoring the activity of these modified glycoproteinhormones, one can determine which modifications yield glycoproteinhormones with reduced activity. These glycoprotein hormone agonists canbe used in studies of the hormone receptor such as receptor turnoverrates, receptor affinity for the glycoprotein hormone, or even intherapeutic procedures such as treatment of Grave's disease and infertility control.

The present invention also provides a method of treating a conditionassociated with a glycoprotein hormone activity in a subject comprisingadministering a therapeutic amount of the glycoprotein hormone of thepresent invention to the subject. These conditions include any conditionassociated with a glycoprotein hormone activity. Examples of theseconditions include, but are not limited to, ovulatory disfunction,luteal phase defect, unexplained infertility, male factor infertility,time-limited conception.

In another example, the glycoprotein hormone may be administered todiagnose and treat a thyroid carcinoma. For example, the administrationof bovine TSH to a human subject can be used to stimulate the uptake of¹³¹I in thyroid tissue to treat thyroid carcinoma. (Meier, C. A., etal., “Diagnostic use of Recombinant Human Thyrotropin in Patients withThyroid Carcinoma (Phase I/II Study).” J. Clin. Endocrinol. Metabol.78:22 (1994)).

A skilled practitioner in the art can readily determine the effectiveamount of the glycoprotein hormone to administer and will depend onfactors such as weight, size, the severity of the specific condition,and the type of subject itself. The therapeutically effective amount canreadily be determined by routine optimization procedures. The presentinvention provides glycoprotein hormones with increased activityrelative to the wild-type glycoprotein hormone. These modifiedglycoprotein hormones will allow a skilled practitioner to administer alower dose of a modified glycoprotein hormone relative to the wild-typeglycoprotein hormones to achieve a similar therapeutic effect, oralternatively, administer a dose of the modified glycoprotein hormonesimilar to the dose of the wild-type glycoprotein hormone to achieve anincreased therapeutic effect.

Depending on whether the glycoprotein hormone is administered orally,parenterally, or otherwise, the administration of the prostaglandin canbe in the form of solid, semi-solid, or liquid dosage forms, such as,for example, tablets, pills, capsules, powders, liquids, creams, andsuspensions, or the like, preferably in unit dosage form suitable fordelivery of a precise dosage. The glycoprotein hormone may include aneffective amount of the selected glycoprotein hormone in combinationwith a pharmaceutically acceptable carrier and, in addition, may includeother medicinal agents, pharmaceutical agents, carriers, adjuvants,diluents, etc. By “pharmaceutically acceptable” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected glycoproteinhormone without causing unacceptable biological effects or interactingin an unacceptable manner with the glycoprotein hormone. Actual methodsof preparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, latest edition (Mack Publishing Co., Easton, Pa.).

In another aspect, the present invention provides a method of assistingreproduction in a subject comprising administering an assisting amountof the glycoprotein hormone of the present invention. For example, in asubject with isolated gonadotropin deficiency (IGD), administration ofmodified follicle stimulating hormone (follitropin) and luteinizinghormone (lutropin) may be administered to the subject to restore normalgonadal function. It is widely known in the art that glycoproteinhormones such as FSH and LH are integral in female reproductivephysiology, and these glycoprotein hormones may be administered to asubject to overcome a number of reproductive disorders and therebyassist reproduction.

Genetic therapy is another approach for treating hormone disorders withthe modified glycoprotein hormones of the present invention. In thisapproach, a gene encoding the modified glycoprotein hormone can beintroduced into a cell, such as a germ line cell or a somatic cell, sothat the gene is expressed in the cell and subsequent generations ofthose cells are capable of expressing the introduced gene. For example,any particular gonadotropin hormone can be inserted into an ovariancell, or its precursor, to enhance ovulation. Alternatively, introducingthyroid cells carrying a gene encoding a superagonist of the thyroidstimulating hormone into an individual with thyroid carcinoma canobviate the need for continual administration of TSH for stimulatingradioiodine uptake in the thyroid carcinoma. Suitable vectors to deliverthe coding sequence are well known in the art. For example, the vectorcould be viral, such as adenoviral, adenoassociated virus, retrovirus,or non-viral, such as cationic liposomes.

The modified glycoprotein hormones as provided by the present inventioncan also be used for targeting delivery of therapeutic agents to thyroidtissues or gonadal tissue, or in the treatment of certain neoplasms.

In yet another aspect, the invention provides a method of constructingsuperactive nonchimeric analogs of human hormones comprising comparingthe amino acid sequence of a more active homolog from another species tothe human hormone, substituting amino acids in the human hormone withthe corresponding amino acids from the other species, determining theactivity of the substituted human hormone, and selecting superactiveanalogs from the substituted human hormones. Superactive analogs ofhuman hormones includes any analog whose activity is increased over thecorresponding activity of the wild-type hormone. For example, themodification of the human thyroid stimulating hormone at position 11 inthe α-subunit from threonine to lysine (T11K) results in a relativeincrease in the cAMP production in JP09 cells cultured in vitro. (SeeTable II as set forth in the Example contained herein). Thismodification of the human thyroid stimulating hormone therefore resultsin a superactive analog of the wild-type human thyroid stimulatinghormone. The specific amino acid or amino acids to substitute to createthe modification can be determined, as discussed above, by: determiningthe activity of the homolog from another species and comparing thatactivity to the human hormone; then comparing the aligned sequences todetermine the amino acid sequence differences; then substituting theappropriate amino acid in the hormone from another species for the aminoacid at the corresponding position in the human hormone; thendetermining the activity of the modified human hormone by one of theabove-mentioned techniques; and then comparing the activity of themodified human hormones to the wild-type human hormone, therebyselecting the superactive analogs from the substituted human hormones.

All combinations of amino acid substitutions may be utilized to obtain aglycoprotein superagonist. For example, neutral amino acids can besubstituted for basic or acidic amino acids. Alternatively, basic aminoacids can be substituted for acidic or neutral amino acids, or acidicamino acids may be substituted for neutral or basic amino acids. Oneskilled in the art will recognize, as discussed above, that substitutionof one amino acid for another can be at either the nucleic acid level inthe nucleotide sequence that encodes the glycoprotein hormone or part ofthe glycoprotein hormone, or at the polypeptide level. Any human hormonecan be modified by this method and its superactive analogs selected. Inparticular, the human hormone can be a glycoprotein hormone.

EXAMPLES

The sequence between Cys10 and Pro21 of the human α-subunit was selectedas the primary target for mutagenesis (FIG. 1). hCG-based homologymodeling suggested that this region of the α-subunit is distant from theβ-subunit in all glycoprotein hormones, contains several surface-exposedresidues and includes a single turn of a 3₁₀-helix between Pro16 andSer19¹. The human α-subunit differs from bovine in position 11, 13, 16,17 and 20 (FIG. 1 a) and four of these changes are nonconservative(Thr11→Lys, Gln13→Lys, Pro16→Lys and Gln20→Lys). We used PCRamplification to determine the sequence of the 11–20 region in theα-subunit of several primates including higher apes (commonchimpanzee—Pan troglodytes, orangutan—Pongo pygmaeus), lesser apes(gibbon—Hylobates sp.), Old World monkey (baboon—Papio anubis) andcompare them with previously known mammalian sequences including. rhesusmacaque (Macaca mulatta; Old World monkey), common marmoset (Callithrixjacchus; New World monkey) and human (FIG. 1 a). Simultaneous comparisonof the sequences between different species suggested that basic residuesin this region were replaced relatively late in primate evolution. TheRhesus monkey α-subunit gene codes for Lys residues at positions 11, 16and 20 and an Arg residue at position 13², the baboon sequence codes forGln at position 16, whereas gibbon sequence contains only one weaklybasic imidazolium group of His at position 13 (FIG. 1 a). Apparently acluster of positively charged amino acids in this region was maintainedand modified during vertebrate evolution, but is not present in thehigher apes and human sequence. The gradual elimination of positivelycharged residues in the 11–20 region of α-subunit coincide with theevolutionary divergence of the hominoids (human and apes) from the OldWorld monkeys. Our hypothesis that this region may modulate binding tothe receptor was further supported by: 1) the highest reactivity ofTyr21 in bTSH toward iodination³, 2) mapping of antigenic determinantsin hCG⁴, 3) the role of amino groups of Lys in the ovine and humanα-subunit for effective hormone-receptor interaction as studied byacylation⁵, labeling with acetic anhydride⁶ and pegylation of individualsubunits.

Consequently, positively charged Lys residues were inserted into theCys10-Pro21 region of the human α-subunit (FIG. 1 b). Two other regionswere also mutagenized (Table I). A single nonconservative Leu69→Argmutation in the TSHβ-subunit was made based on a similar sequencecomparison.

Effects of Mutations

Cotransfection of wild-type (WT) or mutant human α and hTSHβ⁷ or hCGβcDNAs in various combination into CHO-K1 cells resulted in theexpression of 14 hTSH and 11 hCG heterodimers (Table I). In contrast tomany other mutagenesis studies^(8,9) the expression of mutants wasgenerally comparable to the WT. The following hTSH α-mutants wereexpressed at levels higher than WT-hTSH: T11K, Q13K, P16K, Q20K, Q50Pand Q13K+P16K+Q20K. Thus, this set of evolutionary justified mutationsdid not impair, in a major way, synthesis of the hTSH or hCG molecule,but may facilitate in certain cases hormone production.

Various bioassays were used to compare the relative potency and efficacyof hTSH and hCG mutants. The ability of WT and mutant hTSH to stimulatecAMP production was tested in CHO-JP09 cells with stably transfectedhuman TSH receptor. This assay revealed the following order of potenciesin single α-subunit mutants: P16K (6-fold lower EC₅₀ thanWT)≧Q20K>Q13K>T11K>WT-hTSH≈Q50P≈R67K (Table II). Receptor bindingactivity of WT and mutants hTSH was assessed in a competitive bindingassay to porcine thyroid membranes. Consistent with the cAMPstimulation, the following order of potencies was observed: P16K (5-foldgreater affinity than WT)>Q20K≧Q13K>T11K>WT-hTSH≈Q50P≈R67K (Table II).Thus, the increase in potency of single mutants observed in JP09 cellswas directly correlated with the increase of affinity to the TSHreceptor. Most notably, each mutation to a Lys residue in the 11–20region caused a substantial increase in activity, but changes outsidethis critical region had no (R67K, Q50P) effect on receptor bindingaffinity and bioactivity (Table II). Alanine mutagenesis of amino acids13, 16 and 20 in hTSH did not significantly alter hormone activity,indicating that only selective reconstitution of basic amino acidspresent in homologous hormones of other species resulted in thefunctional changes. Moreover, the exchange of αSer43 to Arg and thereplacements of αHis90 and αLys91 showed that these residues were lessimportant for hTSH than for hCG bioactivity, emphasizing hormone- andsite-specific roles of basic residues⁹.

Superagonists with Combined Mutations

To further study the effect of Lys residues which were individuallyresponsible for highest increases in potency, mutants containingmultiple replacements were produced. The most active mutants arepresented in FIGS. 2 and 3. The double Pro16→Lys+Gln20→Lys and thetriple Pro16→Lys+Gln20→Lys+Gln13→Lys mutants showed, respectively, 12and 24-fold higher activity than WT-hTSH, with a further increase inpotency up to 35-fold after Leu69→Arg replacement in the TSHβ-subunit(FIG. 2 a). Additional optimization included substitution Glu14→Lys (Lysin this position present in the tuna sequence) resulted in furtherincrease in bioactivity up to 95-fold; these most potent multiplemutants elevated efficacy (maximal response) at least 1.5-fold (FIG. 2b). These increases were verified by testing the ability of hTSH mutantsto bind to porcine as well as human TSH receptor (Table II, FIG. 2 c andFIG. 2 d), to induce growth in FRTL-5 cells (FIG. 2 e), as well as T₃production in cultured human thyroid follicles. In particular,Pro16→Lys+Gln20→Lys+Gln13→Lys/WT-hTSHβ andPro16→Lys+Gln20→Lys+Gln13→Lys/Leu69→Arg mutants required, respectively,18- and 27-fold lower concentration to attain half-maximal stimulationof ³H-thymidine incorporation in FRTL-5 cells than the WT-hTSH (FIG. 2e). The synergistic effect of multiple mutations on TSH bioactivity wasnot limited to a local cooperation of Lys residues in the 13–20 regionof the α-subunit with receptor, but also involved the contribution ofArg69 in the opposite loop of β-subunit (Table II).

TABLE I Relative expression of wild-type (WT) and mutant hormones inCHO-K1 cells hTSH hCG WT 100 ± 7 100 ± 4 T11K 267 ± 22  82 ± 2 Q13K 188± 9 106 ± 7 P16K 206 ± 25  72 ± 6 Q20K 149 ± 18 117 ± 8 P16K + Q20K  86± 9  62 ± 6 Q13K + P16K + Q20K 134 ± 6  76 ± 12 Q13K + P16K + Q20K +E14K  76 ± 12  52 ± 8 P16K + F17T  23 ± 10  93 ± 4 Q50P 174 ± 15  83 ± 3R67K 171 ± 14  88 ± 6 β3-L69R  74 ± 5 n.a. Q13K + P16K + Q20K + β − L69R 86 ± 6 n.a. Q13K + P16K + Q20K + E14K + β− L69R  25 ± 6 n.a. n.a. = notapplicable. Secretion levels are given as mean ± SEM relative to the WT,which was defined as 100% of WT-hTSH or WT-hCG respectively. The meanwas calculated from at least four independent transfections, performedin at least five dishes for each mutant.These findings were further confirmed in the animal model. A singleinjection of Pro16→Lys, Gln20→Lys and Gln13→Lys hTSH mutants in miceincreased serum T₄ significantly higher than the WT-hTSH. Moreover,Pro16→Lys+Gln20→Lys+Gln13→Lys/WT-hTSHβ andPro16→Lys+Gln20→Lys+Gln13→Lys/Leu69→Arg mutants also generated higher T₄levels as compared to WT-hTSH (FIG. 2 f). hTSH serum levels 6 h afteri.p. injection in mice were similar and the hTSH analogs did not showcompared to the WT great differences in the metabolic clearance rate.

TABLE II The effects of site-specific mutagenesis of human glycoproteinhormones hTSH hTCG cAMP stimulation in JP09 cells Inhibition of¹²⁵I-bTSH Progesterone synthesis in MA10 EC₅₀ (ng/ml) Relative potency(WT = 1) binding (EC₂₅, ng/ml) cells (EC₅₀, ng/ml; Max, %) WT 6.70 ±0.69 1.0 81.3 ± 13.8 6.90 ± 1.04 100 ± 11 T11K 4.47 ± 0.79 1.5 68.3 ±4.4  2.79 ± 0.25 156 ± 23 Q13K 1.89 ± 0.41 3.5 22.5 ± 2.6  2.46 ± 0.28115 ± 24 P16K 1.05 ± 0.26 6.4 18.3 ± 3.6  2.05 ± 0.17 161 ± 31 Q20K 1.16± 0.22 5.8 21.3 ± 3.8  2.98 ± 0.27 134 ± 10 P16K + Q20K 0.57 ± 0.10 11.86.4 ± 2.4 1.70 ± 0.13 212 ± 34 Q13K + P16K + Q20K 0.28 ± 0.07 23.9 2.3 ±0.3 1.58 ± 0.09 216 ± 36 Q13K + P16K + Q20K + E14K 0.17 ± 0.04 39.4 2.1± 0.4 1.65 ± 0.06 205 ± 41 P16K + F17T 3.52 ± 0.50 1.9 n.d. n.d. n.d.Q50P 5.54 ± 0.70 1.2 77.5 ± 12.4 3.90 ± 0.85 137 ± 27 R67K 7.36 ± 0.330.9 62.5 ± 15.5 4.60 ± 0.63 145 ± 12 TSHβ-L69R 2.75 ± 0.49 2.4 n.d. n.a.n.a. TSHβ-L69R + +Q13K + P16K + Q20K 0.19 ± 0.06 35.3 1.8 ± 0.3 n.a.n.a. TSHβ-L69R + +Q13K + P16K + Q20K 0.07 ± 0.02 95.7 1.3 ± 0.4 n.a.n.a. bTSH 0.71 ± 0.14 9.4 7.9 ± 2.5 n.a. n.a. n.s. - no stimulation;n.a. - not applicable; n.d. - not determined. A curve-fitting program,Mac Allfit (NIH, Bethesda, MD) was used to fit the dose-response dataand calculate EC₅₀ and Vmax values. Values are the mean ± SEM.Pro16/Lys + Phe17/Thr mutant with glycosylation consensus sequence(Asn-Lys-Thr) was created to study the effect of neoglycosylation onhormone activity.

A sequence comparison of the hCG and hTSH β-subunits showed a region(residues 58–69 in TSHβ) which contains a cluster of basic residues inhCG, but not in hTSH. We used site-directed mutagenesis to introducesingle and multiple basic residues into hTSH, based on their location inhCG, generating the additional hTSH β-subunit mutants: I58R, E63R,I58R+E63R, 158R+E63R+L69R. The mutant hTSH β-subunits were coexpressedwith the human α-subunit and the intrinsic activity of the recombinanthTSH analogs studied at the rat THS receptor (FRTL-5 cells) and humanTSH receptor (CHO-hTSHr cells). In both systems, single substitutions(I58R, E63R) increased potency of hTSH 2-fold to 4-fold, and led to aslight increase of efficacy (FIG. 2 g). The combination of the twosubstitutions (I58R+E63R) resulted in the potency which was 15-foldhigher than that of wild type hTSH and an 1.5-fold increase of efficacy(FIG. 2 g). Potency and efficacy of the combination mutantI58R+E63R+L69R, in which three basic residues were introduced, waselevated 50-fold and 1.7-fold, respectively (FIG. 2 g). These increasesof intrinsic activity were accompanied by concomitant increases inreceptor binding affinity, judged by a receptor-binding assay usingCHO-JP09 cells. (FIG. 2 h). Similarly, when mice were injected with theI58R+E63R+L69R mutant, their T₄ stimulation was significantly higherthan in either mock or control treated mice. (FIG. 2 i).

The bioactivity of hCG mutants was tested using progesterone stimulationin MA-10 cells and cAMP stimulation in COS-7 cells transfected withhuman LH/hCG receptor. hCG Lys mutants showed both higher potency (lowerEC₅₀ values) as well as higher efficacy (V_(max)) than the WT-hCG (TableII, FIG. 3 a). The effect of single and multiple mutations wasrelatively analogous to that observed for the hTSH mutants. TheαPro16→Lys/WT-hCGβ mutant was 4-fold more active than WT-hCG in thestimulation of progesterone production and receptor binding activity inMA-10 cells, with further increases in both potency and efficacy for thePro16→Lys+Gln20→Lys and Pro16→Lys+Gln20→Lys hCG+Gln13→Lys mutants (FIGS.3 a and 3 b). Similar increases of intrinsic activity were also foundwhen studied at the human LH/hCG receptor (COS-7-hLH/hCG-R cells) (FIGS.3 c and 3 d).

Our data suggest that only a few amino acid replacements are sufficientto increase glycoprotein hormone bioactivity, even to a level higherthan that of the model hormone (such as bTSH). Interestingly, only a fewof the 40 differing residues between bovine and human TSH appearresponsible for the higher biological activity of bTSH. The majority ofthe other replacements are conservative, and as illustrated by the R67Kmutation in the α-subunit, seem to have no functional significance. Incontrast, we show that surface-located Lys residues clustered in the L1loop and β1-strand of the α-subunit are crucial for the high bioactivityof bTSH. Accordingly, recombinant hTSH with only two mutated amino acids(P16K+Q20K) attains an intrinsic activity comparable to bTSH (Table II).Moreover, triple, quadruplicate and quintuple hTSH mutants show evenhigher potency than bTSH. These data suggest that the difference inactivity between bTSH and hTSH is a result of several amino acidchanges, including replacements increasing activity, but also otherswhich may reduce biopotency of bTSH at the hTSH receptor.

Although, we cannot exclude a possibility that several receptor specieswould be made from a single transfected cDNA (by alternative splicingfrom cryptic sites or by posttranslational modifications), the fact thatsimilar differences in activity were observed in different cell systemsargues strongly against the importance of different receptor species inthe increase in potency, efficacy and affinity of these analogs.Furthermore, there is compelling evidence that naturally occurringhormone isoforms with various carbohydrate residues exert their effectat the post-receptor level with no or minimal effect on receptor bindingaffinity¹⁰. Since the wild type hormones and their analogs werecharacterized in multiple experimental systems, it is highly probablethat phenomenon of increased bioactivity described here is a rule ratherthan exception related to particular cell-dependent variant of thereceptor.

Perspectives of Rational Design of Glycoprotein Hormone Analogs

Previous site-directed mutagenesis studies of glycoprotein hormonesfocused primarily on the highly conserved regions and residues, usingsuch strategies as alanine scanning mutagenesis¹¹ or multiplereplacement approaches⁹. Several important studies were based on thecreation of chimeric subunits using cassette mutagenesis and/orrestriction fragment exchange^(12,13,14). Our strategy based onreplacement of nonconserved residues to those present in other specieshas been successful and permitted the generation of other glycoproteinhormone analogs, including hFSH mutants with increased bioactivity. Theparallel improvement of bioactivity of hTSH, hCG and hFSH byintroduction of basic residues in the 11–20 region of human α-subunitmay be related to the fact that this region is distant from theβ-subunit in the crystal structure based model of hCG and in ourhomology model of hTSH. The virtual identity of this area in both modelsas well as the observation that the antibodies binding to 11–26 regionare not greatly influenced by subunit combination¹⁵ suggest that thisdomain may function similarly in all the glycoprotein hormones. Once theα-subunit was successfully engineered to create more potent agonists ofhTSH, hCG or hFSH, the same paradigm was used to modify their respectiveβ-subunits to generate the ultimate superagonists of each glycoproteinhormone. For example, an additional replacement of a nonpolar Leu69 toArg in the TSHβ-subunit resulted in further increase of hTSHbioactivity. In addition, the plasma half-life of our analogs can bemodified regarding to specific therapeutic needs.

Further design and refinement of glycoprotein hormone analogs willinclude detailed three-dimensional structure of the hormone-receptorcomplexes. Although the exact structure of glycoprotein hormonereceptors has not been solved, several models of hormone-receptorinteraction have been proposed^(15,16,17,18). In accordance with therecent model of Jiang et al.¹⁷ the L1 loop of α-subunit may participatein the interaction with the transmembrane portion of the receptor. Thecluster of positively charged residues in this loop may enhance such aninteraction and facilitate further rearrangements in the receptorleading to the activation of G proteins and signal transduction.

Methods and Materials.

Restriction enzymes, DNA markers and other molecular biological reagentswere purchased from either Gibco BRL (Gaithersburg, Md.) or fromBoehringer-Mannheim (Indianapolis, Ind.). Cell culture media, fetalbovine serum and LipofectAMINE were purchased from Gibco BRL(Gaithersburg, Md.). VentR DNA Polymerase was purchased from New EnglandBiolabs (Beverly, Mass.). The full length human α cDNA (840 bp)subcloned into BamHI/XhoI sites of the pcDNA I/Neo vector (InvitrogenCorp., San Diego, Calif.) and hCG-β gene were obtained from Dr. T. H. Ji(University of Wyoming, Laramie, Wash.). The hTSH-β minigene without thefirst intron, with the non-translated 1st exon and authentic translationinitiation site was constructed in our laboratory. rhTSH-G standard wasfrom Genzyme Corp. (Framingham, Mass.). The CHO cells with stablyexpressed hTSH receptor (CHO-hTSHR clone JP09 and clone JP26) wereprovided by Dr. G. Vassart (University of Brussels, Brussels, Belgium).The human LH receptor cDNA was obtained from Dr. T. Minegishi (GunmaUniversity, Gunma, Japan). FRTL-5 cells were kindly supplied by Dr. L.D. Kohn (NIDDK, NIH, Bethesda, Md.). MA-10 cells were generouslysupplied by Dr. M. Ascoli (University of Iowa, Iowa City, Iowa). ¹²⁵IcAMP and ¹²⁵I-hTSH were from Hazleton Biologicals (Vienna, Va.). Bloodsamples of various primates were obtained from Yerkes Regional PrimateResearch Center (Emory University, Atlanta, Ga.) and Animal Resources(University of Oklahoma, Oklahoma City, Okla.).

Determination of Primate α-Subunit Sequences

The QIAamp^(R) Blood Kit (Qiagen Inc., Chatsworth, Calif.) was used forextraction of genomic DNA from whole blood samples of chimpanzee (Pantroglodytes), orangutan (Pongo pygmaeus), gibbon (Hylobates sp.) andbaboon (Papio anubis). Genomic DNA was used in the PCR; the syntheticoligonucleotide primers used were

5′-CCTGATAGATTGCCCAGAATGC-3′ (sense) (SEQ ID NO:1) and

5′-GTGATAATAACAAGTACTGCAGTG-3′ (antisense) (SEQ ID NO:2) and weresynthesized according to the nucleotide sequence of the gene encodingcommon α-subunit of human glycoprotein hormones¹⁹. PCR was performedusing 800–1000 ng of genomic DNA template and 10 picomoles of eachprimer in 100 μl reaction volume that also contained 10 mM Tris-HCl, (pH9.0 at 25° C.), 50 mM KCl, 2.5 mM MgCl₂, 200 μM dNTPs and 2 U of Taq DNAPolymerase (Promega Corp. Madison, Wis.). The reaction mix was coveredwith mineral oil, and each sample was initially heated to 95° C. for 10min. The PCR program consisted of 32 cycles of denaturation at 95° C.for 1 min 30 sec, annealing at 55° C. for 1 min 30 sec and extension at72° C. for 1 min, followed by a final extension period at 72° C. for 7min. The reactions were then directly electrophoresed on a 1% agarosegel in the presence of ethidium bromide. The amplified PCR product (˜700bp), spanning the nucleotide sequence of exon 3, intron 3 and exon 4,was purified using QIAquick PCR Purification Kit (QIAGEN Inc.,Chatsworth, Calif.) and subcloned into pCR™II using Original TA CloningKit (Invitrogen Corp., San Diego, Calif.). The sequence of the fragmentwas obtained after subcloning or direct dideoxy sequencing using aSequenase kit (U.S. Biochemical Corp., Cleveland, Ohio).

Homology Modeling.

Modeling relies on the strong sequence homology between hCG and hTSH.The sequences were aligned to bring the cysteine-knot residues intocorrespondence and the percentage of identical as well as highlyconservative replacements were calculated as described¹. There was 58%sequence identity between hCG and hTSH molecules; 31% of the twoβ-subunit sequences were identical and additional 17% included highlyconservative changes in β-subunit. A molecular model of hTSH was builton a template of hCG model derived from crystallographic coordinatesobtained from the Brookhaven Data Bank²⁰. All coordinate manipulationsand energy calculations were done using CHARMm release 21.2 for theConvex and further modified using the molecular graphic package QUANTA(Version 3.3, Molecular Simulations Inc., University of York, York,United Kingdom).

Site-Directed Mutagenesis.

Mutagenesis of the human α-cDNA and the hTSHβ minigene was accomplishedby the PCR-based megaprimer method²¹. Amplification was optimized usingVent^(R) DNA Polymerase (New England Biolabs, Beverly, Mass.). Afterdigestion with BamH1 and Xho1 PCR product was ligated into pcDNA I/Neo(Invitrogen Corp., San Diego, Calif.) with the BamHI/XhoI fragmentexcised. MC1061/p3 E. coli cells were transformed using Ultracomp E.coli Transformation Kit (Invitrogen Corp.). The QIAprep 8 Plasmid Kit(QIAGEN Inc., Chatsworth, Calif.) was used for multiple plasmid DNApreparations. QIAGEN Mega and Maxi Purification Protocols were used topurify larger quantities of plasmid DNA. Multiple mutants were createdwith the same method using plasmids containing α-cDNA with a singlemutation as a template for further mutagenesis. Mutations were confirmedby double stranded sequencing using Sanger's dideoxynucleotide chaintermination procedure.

Expression of Recombinant Hormones.

CHO-K1 Cells (ATCC, Rockville, Md.) were maintained in Ham's F-12 mediumwith glutamine and 10% FBS, penicillin (50 units/mil) and streptomycin(50 μg/ml). Plates of cells (100 mm culture dishes) were cotransfectedwith wild type or mutant α-cDNA in the pcDNAI/NEO and hTSHβ minigeneinserted into the p(LB)CMV vector⁷, or pcDNAI/Neo containing hCGβ-cDNA⁸using a LipofectAMINE (Gibco BRL, Gaithersburg, Md.). After 24 h, thetransfected cells were transferred to CHO-serum free medium (CHO-SFM-II,Gibco BRL,). The culture media including control medium from mocktransfections using the expression plasmids without gene inserts wereharvested 72 h after transfection, concentrated and centrifuged; thealiquots were stored at −20° C. and thawed only once before each assay.WT and mutant hTSH were measured and verified using four differentimmunoassays as described⁹. Concentrations of WT and mutant hCG weremeasured using chemiluminescence assay (hCG Kit, Nichols Institute, SanJuan Capistrano, Calif.) and immunoradiometric assay (hCG IRMA, ICN,Costa Mesa, Calif.).

cAMP Stimulation in JP09 Cells Expressing Human TSH Receptor.

CHO cells stably transfected with hTSH receptor cDNA (JP09) were grownand incubated with serial dilutions of WT and mutant hTSH as described⁹.cAMP released into the medium was measured by radioimmunoassay²². Theequivalent amounts of total media protein were used as the mock controland the hTSH containing samples from transfected cells.

cAMP Stimulation in COS-7 Cells Expressing Human LH Receptor.

COS-7 cells transiently transfected with hLH receptor cDNA were grownand incubated with serial dilutions of WT and mutant hCG essentially asdescribed²³. cAMP released into the medium was measured byradioimmunoassay²². The equivalent amounts of total media protein wereused as the mock control and the hCG containing samples from transfectedcells.

Progesterone Production Stimulation in MA-10 Cells.

Transformed murine Leydig cells (MA-10) grown in 96-well culture plateswere incubated with WT and mutant hCG for 6 hours in the assay medium asdescribed²⁴. The amount of progesterone released into the medium wasdetermined by radioimmunoassay (CT Progesterone Kit, ICN Biomedicals,Inc., Costa Mesa, Calif.).

Receptor Binding Assays.

The receptor-binding activities of hTSH analogs were assayed by theirability to displace ¹²⁵I-bTSH from a solubilized porcine thyroidmembranes²²⁴. The binding activities of selected analogs to human TSHreceptor was tested using JP09 cells. The binding activities of hCGanalogs to MA-cells and to COS-7 cells transiently transfected withhuman LH receptor were determined using ¹²⁵I-hCG and assay medium asdescribed previously²⁴.

Thymidine Uptake Stimulation in FRTL-5 Cells.

Growth of the rat thyroid cells (FRTL-5) was monitored as previouslydescribed²².

Stimulation of T₄ Secretion in Mice.

The in vivo bioactivity of the WT and mutant TSH was determined using amodified McKenzie bioassay^(22,25). WT and mutant TSH were injected i.p.into male albino Swiss Cr1:CF-1 mice with previously suppressedendogenous TSH by administration of 3 μg/ml T₃ in drinking water for 6days. Blood samples were collected 6 h later from orbital sinus and theserum T₄ and TSH levels were measured by respective chemiluminescenceassays (Nichols Institute).

Throughout this application various publications are referenced. Certainpublications are referenced by numbers within parentheses. Fullcitations for the number-referenced publications are listed below. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

REFERENCES

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1. A modified human follicle stimulating hormone (FSH), which differsfrom the wild-type human FSH, said modified human FSH comprising anα-subunit and a β-subunit, said α-subunit comprising at least threebasic amino acids in the α-subunit at positions selected from the groupconsisting of positions 11, 13, 14, 16, 17, and 20, wherein by human ismeant the number of amino acid substitutions in the wild-type sequencedoes not exceed one-half the number of amino acid differences atcorresponding positions in the FSH subunits between human and bovinespecies.
 2. The modified human FSH of claim 1, said α-subunit furthercomprising a fourth basic amino acid at a position selected from thegroup consisting of positions 11, 13, 14, 16, 17, and
 20. 3. Themodified human FSH of claim 2, wherein said basic amino acids of theα-subunit are at positions 11, 13, 16, and
 20. 4. The modified human FSHof claim 2, wherein said basic amino acids of the α-subunit are atpositions 11, 13, 17, and
 20. 5. The modified human FSH of claim 2,wherein said basic amino acids of the α-subunit are at positions 13, 14,16, and
 20. 6. The modified human FSH of claim 2, wherein said basicamino acids of the α-subunit are at positions 13, 14, 17, and
 20. 7. Themodified human FSH of claim 2, said α-subunit further comprising a fifthbasic amino acid at a position selected from the group consisting ofpositions 11, 13, 14, 16, 17, and
 20. 8. The modified human FSH of claim7, wherein said basic amino acids of the α-subunit are at positions 13,14, 16, 17, and
 20. 9. The modified human FSH of claim 7, wherein saidbasic amino acids of the α-subunit are at positions 11, 13, 14, 16, and20.
 10. The modified human FSH of claim 1, wherein said basic aminoacids of the α-subunit are at positions 11, 13, 14, 16, 17, and
 20. 11.The modified human FSH of claim 1, wherein said basic amino acids of theα-subunit are at positions 13, 16, and
 20. 12. The modified human FSH ofclaim 1, wherein said basic amino acids of the α-subunit are atpositions 14, 16, and
 20. 13. The modified human FSH of claim 1, whereinsaid basic amino acids are selected from the group consisting of lysineand arginine.
 14. A nucleic acid encoding the modified human FSHα-subunit of claim
 1. 15. A vector comprising the nucleic acid of claim14, wherein the vector is suitable for expressing the nucleic acid. 16.A host cell comprising the vector of claim 15, wherein the host cell issuitable for expressing the nucleic acid.
 17. The modified human FSH ofclaim 1, wherein said modified human FSH has less than five amino acidsubstitutions in said α-subunit in positions other than positions 11,13, 14, 16, 17, and
 20. 18. The modified human FSH of claim 1, whereinsaid modified human FSH has less than four amino acid substitutions insaid α-subunit in positions other than positions 11, 13, 14, 16, 17, and20.
 19. The modified human FSH of claim 1, wherein said modified humanFSH has less than three amino acid substitutions in said α-subunit inpositions other than positions 11, 13, 14, 16, 17, and
 20. 20. Themodified human FSH of claim 1, wherein said modified human FSH has lessthan two amino acid substitutions in said α-subunit in positions otherthan positions 11, 13, 14, 16, 17, and
 20. 21. The modified human FSH ofclaim 1, wherein said modified human FSH has complete amino acidsequence identity with the corresponding wild-type human FSH in saidα-subunit in positions other than positions 11, 13, 14, 16, 17, and 20.22. A modified human follicle stimulating hormone (FSH), which differsfrom the wild-type human FSH, said modified human FSH comprising anα-subunit and a β-subunit, said α-subunit comprising a basic amino acidin the α-subunit in at least one position selected from the groupconsisting of positions 11, 13, 14, 16, 17, and 20, wherein by human ismeant the number of amino acid substitutions in the wild-type sequencedoes not exceed one-half the number of amino acid differences atcorresponding positions in the FSH subunits between human and bovinespecies.
 23. The modified human FSH of claim 22, wherein a basic aminoacid of the α-subunit is at position
 11. 24. The modified human FSH ofclaim 22, wherein a basic amino acid of the α-subunit is at position 13.25. The modified human FSH of claim 22, wherein a basic amino acid ofthe α-subunit is at position
 14. 26. The modified human FSH of claim 22,wherein a basic amino acid of the α-subunit is at position
 16. 27. Themodified human FSH of claim 22, wherein a basic amino acid of theα-subunit is at position
 17. 28. The modified human FSH of claim 22,wherein a basic amino acid of the α-subunit is at position
 20. 29. Themodified human FSH of claim 22, wherein said basic amino acid isselected from the group consisting of lysine and arginine.
 30. Themodified human FSH of claim 22, further modified so that said α-subunitcomprises a basic amino acid in at least two positions selected from thegroup consisting of positions 11, 13, 14, 16, 17, and
 20. 31. Themodified FSH of claim 30, wherein said basic amino acids of theα-subunit are at positions 14 and
 20. 32. The modified human FSH ofclaim 30, wherein said basic amino acids of the α-subunit are atpositions 16 and
 20. 33. The modified human FSH of claim 30, whereinsaid basic amino acids of the α-subunit are at positions 13 and
 14. 34.The modified human FSH of claim 30, wherein said basic amino acids ofthe α-subunit are at positions 13 and
 16. 35. The modified human FSH ofclaim 30, wherein said basic amino acids of the α-subunit are atpositions 13 and
 20. 36. The modified human FSH of claim 30 wherein saidbasic amino acids of the α-subunit are at positions 14 and
 16. 37. Themodified human FSH of claim 30, wherein said basic amino acid isselected from the group consisting of lysine and arginine.
 38. A nucleicacid encoding the modified human FSH α-subunit of claim
 22. 39. A vectorcomprising the nucleic acid of claim 38, wherein the vector is suitablefor expressing the nucleic acid.
 40. A host cell comprising the vectorof claim 39, wherein the host cell is suitable for expressing thenucleic acid.
 41. The modified human FSH of claim 22, wherein saidmodified human FSH has less than five amino acid substitutions in saidα-subunit in positions other than positions 11, 13, 14, 16, 17, and20.42. The modified human FSH of claim 22, wherein said modified human FSHhas less than four amino acid substitutions in said α-subunit inpositions other than positions 11, 13, 14, 16, 17, and
 20. 43. Themodified human FSH of claim 22, wherein said modified human FSH has lessthan three amino acid substitutions in said α-subunit in positions otherthan positions 11, 13, 14, 16, 17, and20.
 44. The modified human FSH ofclaim 22, wherein said modified human FSH has less than two amino acidsubstitutions in said α-subunit in positions other than positions 11,13, 14, 16, 17, and
 20. 45. The modified human FSH of claim 22, whereinsaid modified human FSH has complete amino acid sequence identity withthe corresponding wild-type human FSH in said α-subunit in positionsother than positions 11, 13, 14, 16, 17, and 20.